The Draize eye irritation test has been in use since 1944. One eye of a conscious rabbit is instilled with the test substance, while the other eye is the control. The reactions of the cornea, conjunctiva, and iris are then evaluated in a period of one hour to three weeks. Because of the test's moral and scientific short comings, a replacement has been actively sought, but there is still none in the horizon. Today, the Draize test remains the only one that can classify a substance into one of the four categories of eye irritation: corrosive/severe, moderate, mild, and non- irritants. Specifically, none of the in viro methods can identify the moderate and mild irritants and the Draize test continues. None of the in vitro method addresses the issue of ocular inflammation, an integral part of the Draize eye irritation test brought upon by dying cells following test substance instillation. We hypothesize that an in vitro method with an end point that can predict the inflammatory potential of the injured eye can identify moderate and mild irritants, in addition to the corrosive/severe and non- irritants. Using human donor corneas not used for transplantation, our preliminary studies demonstrated in a totally animal (and animal product)-free system that the levels of corneal endothelial damage, revealed by alizarin red S staining and quantified by image analysis of the red stain in the panoramic view of the corneal endothelium, predicted with 100% accuracy the irritation potential of 18 test substances in the corrosive/severe, moderate, mild, and non- irritants categories. Our goal is to demonstrate that our method can fully replace the Draize eye irritation test. The specific aims are to 1) use our method to classify test substances that have historical Draize test data available in public, 2) study the status of VIP in the damaged corneal endothelium in the test substance-treated human donor corneas.